Project Summary Mycobacterium tuberculosis is a human-specific pathogen and the etiological agent of the respiratory disease tuberculosis (TB). This bacterium is responsible for significant morbidity and mortality worldwide, producing more than 10 million new cases of active TB disease, and killing more than 1.7 million individuals each year. A key aspect of the M. tuberculosis lifecycle is the ability to produce intracytoplasmic lipid droplets (LDs) and enter into an altered physiological state of non-replicating persistence (NRP). These processes are critical for long-term survival of M. tuberculosis during latency. LDs are quasi-organelles comprised of a neutral fatty acid core surrounded by a phospholipid monolayer and proteins. It is thought that the primary role for LDs in Mycobacterium is to store the neutral fatty acid triacylglycerol (TAG) for use in energy-generating pathways during latency. However, preliminary data described in this application indicates that Mycobacterium LDs may carry out additional functions. In particular, we have demonstrated that Mycobacterium LDs contain an unusually large number of proteins that participate in active cellular processes at the plasma membrane or in the cytoplasmic compartment including ATP production, transcription and translation, and DNA replication. Furthermore, Mycobacterium LDs harbor numerous DNA-binding proteins, including HupB, which is an HU-like DNA-binding protein capable of compacting genomic DNA into condensed DNA nucleoids. In this application, a novel LD culture model and a surrogate Mycobacterium smegmatis host will be used to test the hypothesis that LDs directly regulate the transition of Mycobacterium into NRP. This hypothesis will be tested by determining if Mycobacterium LDs: (i) sequester proteins that normally function in key cellular processes occurring in the cytoplasm or at the plasma membrane (Aim 1), and (ii) bind and compact genomic DNA into condensed DNA nucleoids that are inaccessible for DNA replication (Aim 2). If one or both of these activities are validated, this information is expected to be paradigm changing and will greatly enhance our understanding of M. tuberculosis physiology during NRP within the host.